ClinVar Genomic variation as it relates to human health

Variant summary: LDLR c.1061A>T (p.Asp354Val) results in a non-conservative amino acid change located in the EGF-like domain of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. Other variants have been reported as pathogenic or likley pathogenic in the same codon, suggesting a functional role of the codon (c.1061A>G, p.Asp354Gly; c.1061A>C, pAsp354Ala)The variant allele was found at a frequency of 6.5e-05 in 30944 control chromosomes. c.1061A>T has been reported in the literature in an individual affected with Familial Hypercholesterolemia who had reduced LDLR activity (Hobbs_1992). Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as likely pathogenic. Based on the evidence outlined above, the variant was classified as likely pathogenic.

This missense variant (also known as p.Asp333Val in the mature protein) is located in the EGF-like repeat A in the EGF precursor homology domain of the LDLR protein. Computational prediction tools and conservation analyses suggest that this variant may have deleterious impact on the protein function. Computational splicing tools suggest that this variant may not impact the RNA splicing. Experimental functional assays have shown that this variant significantly reduces LDLR activity in cells from a heterozygous patient (PMID: 1301956). This variant has been reported in individuals affected with familial hypercholesterolemia (PMID: 1301956, 17142622, 19837725). This variant has been identified in 2/30944 chromosomes (2/14980 non-Finnish European chromosomes) in the general population by the Genome Aggregation Database (gnomAD). Based on available evidence, this variant is classified as Likely Pathogenic.

This sequence change replaces aspartic acid, which is acidic and polar, with valine, which is neutral and non-polar, at codon 354 of the LDLR protein (p.Asp354Val). This variant is present in population databases (no rsID available, gnomAD 0.01%). This missense change has been observed in individuals with familial hypercholesterolemia (PMID: 1301956, 19837725). This variant is also known as Asp333Val. ClinVar contains an entry for this variant (Variation ID: 251640). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. This variant disrupts the p.Asp354 amino acid residue in LDLR. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 20145306, 20809525, 22698793). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

The p.D354V variant (also known as c.1061A>T) is located in coding exon 8 of the LDLR gene. The aspartic acid at codon 354 is replaced by valine, an amino acid with highly dissimilar properties. This change occurs in the first base pair of coding exon 8. This variant (also referred to as p.D333V) has been detected in individuals and cohorts with familial hypercholesterolemia (FH), and in individuals undergoing genetic testing for suspicion of FH (Sozen M et al. Atheroscler Suppl, 2004 Dec;5:7-11; Humphries SE et al. J Med Genet, 2006 Dec;43:943-9; Brown EE et al. J Clin Lipidol, 2020 Mar;14:331-338; Sturm AC et al. JAMA Cardiol, 2021 Aug;6:902-909; Ambry internal data). This variant was detected in an individual from an FH cohort demonstrating 15-30% LDLR activity who was indicated as being compound heterozygous for another, unidentified allele (Hobbs HH et al. Hum Mutat, 1992;1:445-66). Another alteration at the same codon, p.D354G (c.1061A>G, also referred to as p.D333G), has also been reported in association with FH (Mosig S et al. BMC Med Genomics. 2008 Nov;1:60). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the majority of available evidence to date, this variant is likely to be pathogenic.

Identified in association with hypercholesterolemia and early-onset myocardial infarction in published literature and in ClinVar (PMID: 32220565, 1301956, 15556093, 19837725, 30586733, 32041611, 34037665); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Also known as p.(D333V) and FH Oklahoma; This variant is associated with the following publications: (PMID: 17142622, 1301956, 32041611, 15556093, 30586733, 34037665, 19837725, 32220565)

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with familial hypercholesterolemia (MIM#143890) and LDL cholesterol level QTL2 (MIM#143890). (I) 0108 - This gene is associated with both recessive and dominant disease. Biallelic individuals have an earlier and more severe onset (GeneReviews). (I) 0112 - The condition associated with this gene has incomplete penetrance (GeneReviews). (I) 0200 - Variant is predicted to result in a missense amino acid change from aspartic acid to valine. (I) 0251 - This variant is heterozygous. (I) 0302 - Variant is present in gnomAD (v3) <0.001 for a dominant condition (2 heterozygotes, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v3) (1 heterozygote, 0 homozygotes). (I) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0600 - Variant is located in the annotated calcium-binding EGF-like domain (NCBI). (I) 0702 - Other missense variants comparable to the one identified in this case have strong previous evidence for pathogenicity. Three alternative changes at the same codon have been reported in multiple individuals with familial hypercholesterolemia (ClinVar, PMID: 29353225). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported in multiple individuals with familial hypercholesterolemia (ClinVar, PMID: 1301956). (SP) 0905 - No published segregation evidence has been identified for this variant. (I) 1001 - This variant has strong functional evidence supporting abnormal protein function. Functional studies using fibroblast has demonstrated reduced LDL receptor activity (PMID: 1301956). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

PP3, PP4, PM2_moderate, PM5, PS3_supporting, PS4_supporting

Genetic testing: The patient had genetic testing for the familial hypercholesterolemia panel. The test included sequencing of three genes associated with familial hypercholesterolemia: LDLR, APOB, PCSK9 and LDLRAP1. Results showed that the following variant was identified (see report below): p.Asp354Val (c.1061A>T) in the LDLR gene (NM_000527.4) The lab classifies this variant as pathogenic. Given case data, functional studies and other pathogenic variants at this same amino acid (p.Asp354Gly and p.Asp354Ala) we consider this variant likely pathogenic and we feel it is suitable for assessing risk in healthy relatives ("predictive genetic testing"). The variant has been seen in at least four unrelated cases of FH (not including this patient's family). There is good case data. This variant is historically known as p.Asp333Val. Hobbs, et al., 1992 (PMID: 1301956) reported this variant in a patient of American indian background and had 15-30 % of receptor activity. They classify this variant as a class 5, recycling defect allele. These bind and internalize ligands in coated pits, but fail to release the ligands in the endosome and thus do not recycle to the cell surface. They classify this variant as FH Oklahoma. Wittall, et al., 2010 (PMID: 19837725) reported this in one of their Greek patients with FH. Invitae has also seen this variant in multiple patients with FH. In silico analysis with PolyPhen-2 predicts the variant to be probably damaging (HumVar: 1.000). The Asp at codon 354 is conserved across species, as are neighboring amino acids. This variant is the first amino acid of the EGF-like 2 calcium binding domain. There is one individual with variation at codon 354 listed in the Genome Aggregation Consortium Dataset (gnomAD; http://gnomad.broadinstitute.org/), which currently includes variant calls on >140,000 unrelated individuals of African, Asian, European, Latino, and Ashkenazi descent. This indicates that variants at this position are rare.